Technical Abstract:
Immunogenic, pathogen-specific proteins have excellent potential for development of novel management modalities. Here, we describe an innovative application of proteomics called Microbial protein-Antigenome Determination (MAD) Technology for rapid identification of native microbial proteins that elicit and interact with host humoral immune responses. To demonstrate “proof of principle”, we cultured the foodborne pathogen E. coli O157:H7 (O157), in vitro, in minimal medium supplemented with norepinephrine (NE), a Beta-adrenergic hormone found in the mammalian gastrointestinal tract, and a mimic of auto-inducer 3 that regulates expression of O157 proteins via quorum sensing. NE-induced O157 proteins were immunoaffinity captured using polyclonal antibodies from pooled hyperimmune sera of cattle, and defined by one dimensional SDS-PAGE liquid chromatography tandem mass spectrometry (GeLC-MS/MS), followed by SEQUEST database searching. MAD identified 91 O157 proteins with genome-wide distribution, as being part of the antigenome in bovine reservoirs, including previously identified O157 adhesins, O157 proteins identified using other proteomics-based approaches such as Proteomics-based Expression Library Screening (PELS; Kudva et al., Mol. Cell. Proteomics 5:1514-1519, 2006); and a subgroup O157 proteins expressed specifically during human infection (John et al., Infect. Immun., 73:2665-2679, 2005). Additionally, a subset of novel extracytoplasmic O157 proteins, unique to this study, was identified. Currently, the adherence potential of a number of these novel proteins is being systematically evaluated, as a prelude to future experiments in cattle. The MAD technology is an innovative, rapid proteomics-based platform application for determining antigenomes expressed by microbial pathogens cultured in vitro under conditions that approximate the host environment. MAD can be applied to any sequenced pathogen capable of eliciting host humoral immune responses, and can complement other strategies for proteome-wide identification of immunogenic microbial proteins.